Characteristics Of Physiology And Expression Profiling Of Soybean Roots Under Aluminum Toxicity And Functional Study Of The MATE Gene Related To Aluminum Tolerance

As the most abundant mental element in earth,aluminum(Al),is a main inorganic mineral in soil.The soluble Al3+ in acidic soils(pH<5.0)mainly inhibits the root elongation even at low concentrations,which subsequently prevents the uptake and translocation of water and nutrients of roots.Al3+induces the production of reactive oxygen spcies and lipid peroxidation,and influences the movement of substances from cell to cell by callose deposition in apoplast.Therefore,Aluminum toxicity,mainly due to the existence of phytotoxic species of Al(Al3+),is an important restraint to soybean production.Limited information is known about Al-tolerant genes and mechanism in soybean.The identification and functional study of Al tolerant genes and their functions,as well as the study of the molecular network regulating Al tolerance in soybean,will provide an important theoretical basis for soybean molecular breedingfor Al tolerance.In the study,we identified the metabolic pathway and candidate genes that might be involved in Al tolerance by expression profiling of Al-tolerant(KF)and Al-sensitive(GF)soybean varieties using microarray.The gene expression patterns of microarray were verified by qRT-PCR.The functions of Al-tolerance candidate gene GmMATE75 was studied by transgenic Arabidopsis and soybeanhairy roots.The main results of this study were as follows:1.Effects of Aluminum toxicity stress on different sensitive soybean varietiesSoybean varieties of KF and GF were identified as Al-tolerant and Al-sensitive at seedlingstage in our previous study.In this study,we investigated the effect of long term Al toxicity on the biomass and physiology in soybean.When the concentration of AlCl3is20mM,the biomass(root dry weight and shoot dry weight)and plant height of GF wassignificantly inhibited.The activies of anti-oxidation enzymes,including POD(peroxidase),SOD(Superoxide Dismutase),and CAT(Catalase),increased by A1 in both varieties,but more significantly in KF than GF.The membrane permeability indicator,MDA,and the conductivity of leaves and roots increased significantly in GF.These results implied that there was no obvious influence on the growth of soybean at low concentration of AICl3,but significantly affected the physiology and growth of Al-sensitive variety GF.While the Al-tolerant variety KF was able to maintain its relatively normal growth by increasing the activities of anti-oxidation enzymes.2.Expression profiling analysis of soybean roots under Al stressThe genome-wide gene expression profiling of KF and GF roots under 0 and 25 μM AICl3(pH 4.3)at 6,12,and 24 hwas investigated usingthe Affymatrix microarray.The differentially expressed genes(DEGs)were identified by the threshold ofFDR(False Discovery Rate)≤0.05 and |log2FC(Fold Change)|≥1.The numbers of up-or down-regulated genes under Al stress increased with the duration of treatment time.The Al-sensitive cultivar GF displayed a higher number of up-/down-regulated genes compared with Al-tolerant cultivar KF.MapMan analysis showed genes involved in themetabolic pathways of cell wall,lipids,glycolysis,tricarboxylic acid cycle and organic acid relatedwere differently regulated between Al-tolerant and Al-sensitive soybean varieties.A total of 1937 genes showed significantly up-regulation by Al in KF compared with GF,which were significantly enriched in antioxidant enzymes by AgriGO analysis.QRT-PCR analysis validated that the POD gene expression pattern as observed in microarray.ThePOD activity increased significantly after Al stress in KFcompared with GF.Gene Ontology(GO)annotationshowed that DEGs were annotated into 26 functional categories.The WRKY,MYB,AP2 transcription factors were also enriched in these 1937 genes which showed significant up-regulation in KF.Our results suggest that the genes related to antioxidant enzymes,WRKY,MYB,AP2transcription factors,and extrusion of organic compunds might be important for Al tolerance in soybean.3.Genome-wide analysis and bioinformatic analysis of MATE transporters in soybeanMultidrug and toxic compound extrusion(MATE)family is an important group of the multidrug effluxtransporters that extrude organic compounds,transporting a broad range of substrates such as organic acids,planthormones and secondary metabolites.However,genome-wide analysis of MATE family in plant species is limitedand no such studies have been reported in soybean.Based on the sequence homology to Arabidopsis MATE and HMMER index,117GmMATE genes were identified from the soybean genome,and we named them as GmMATEl-GmMATE117 which located in 20 chromosomeson the basis ofphysical location.Bioinformatics analysis demonstrated that the GmMATE family could be classified into four groups comprising ten smaller subgroups.Genetic structure and conserved motif analysis revealed that each gene of GmMATEcontained highly conserved motifs.Experiments were carried out to confirm the expression levels of the genes of each group in nine tissues of soybean using qRT-PCR.The results revealed that the expression pattern of these genes were quite different and genes in the same family were also different.Eight soybean MATE transporters clustered together with the previously reported MATE proteins related toAldetoxification and iron translocation were further analyzed.Using PlantCARE databases,seven stress-responsive cis-elements such as ABRE,ARE,HSE,LTR,MBS,as well as a cis-element of ART1(Al resistance transcription factor 1),GGNVS,were identifiedin the upstream region of these eight GmMATE genes.Differential gene expression analysis of these eight GmMATEgenes in response to A1 stress helps us identify GmMATE75 as the candidate gene for Al tolerance in soybean,whoserelative transcript abundance increased at 6,12 and 24 h after Al treatment,with more fold changes in Al-tolerant thanAl-sensitive cultivar,which is consistent withpreviously reported Al-tolerance related MATE genes.4.The functional research of MATE related to Al in soybeanThe coding sequence of GmMATE75 was cloned from KF and overexpressed in Arabidopsiswild type(Col-0)and soybean Al-sensitive GF hairy roots.The accumulation of Al(determined by hematoxylin staining)and ROS in the roots ofGmMATE75 overexpressing(OE)Arabidopsis decreased significantly compared to the WT and mutant.And the root length of OE Arabidopsis was also significantly longer than WT under Al stress.The Al concentration in GmMATE75overexpressing soybean hairy roots was also significantly lower than that of control(hairy roots with empty vector).The resultssuggest that GmMATE75 plays a role in soybean tolerance to Al toxcity.